The immune responses induced by vaccination or natural infection fail to effectively prevent and control HIV infection. Thus, it is important to explore alternative immunization approaches and novel adjuvants to generate protective immune response that is superior to the natural immunity against HIV infection. Antigen-presenting cells (APCs) such as dendritic cells (DCs) play a critical role in the activation and maintenance of immune responses, and they are regulated by stimulatory as well as inhibitory signaling. Recently, we found that the negative regulators of proinflammatory signaling, such as the zinc-finger ubiquitin-modifying enzyme A20 and suppressor of cytokine signaling 1 (SOCS1), play critical roles in limiting the immunostimulatory potency of APCs and the autoreactive response against self-antigens. We demonstrated that silencing of A20 or SOCS1 drastically enhanced the stimulatory potency of TLR agonists and DC vaccines to induce both T cell and antibody responses. In this study we aim to develop novel and potent adjuvants by inhibiting key negative regulators of proinflammatory signaling to facilitate the development of prophylactic and therapeutic vaccines against HIV. The central hypothesis of this study is that a new type of vaccine adjuvants comprised of a TLR ligand and an inhibitor of the negative regulator of proinflammatory signaling for triggering and sustaining TLR signaling cascades in APCs can be created to enhance the magnitude and duration of cellular and humoral responses against HIV to higher levels that cannot be achieved by currently described vaccination approaches or natural infection and may be capable of overcoming HIV's immune evasion and suppression. The specific aims of this study are: 1). To test whether soluble flagellin (FliC)-protamine (P) fusion protein/siA20 complexes can potently stimulate APCs as novel adjuvants to induce stronger and broader HIV-specific CTL and Th responses in mice; 2). To investigate whether immunization of FliC-P/siA20 complexes and mutated HIV Env with enhanced exposure of naturally shielded, protective epitopes more efficiently induces neutralizing antibodies against HIV in mice and rabbits; and 3). To test whether FliC-P/sihA20 complexes are superior to TLR agonists in activating human DCs to stimulate HIV-specific T and B cells for potential clinic use. In summary, there is a compelling need for the development of the novel adjuvant comprised of TLR ligands and A20 inhibitors that have a unique stimulatory ability to enhance innate and adaptive cellular and humoral responses against HIV to higher levels that cannot be achieved by currently described adjuvants. In this proposed study, we aim to develop novel adjuvants for HIV vaccination to induce protective cellular and humoral immune responses. Specifically, we will generate and test the novel adjuvant comprised of TLR ligands and A20 inhibitors that have a unique stimulatory ability to enhance innate and adaptive cellular and humoral responses against HIV to higher levels that cannot be achieved by currently described adjuvants.